Process for production of viomycin



United States Patent 9 I 2,828,245 PROCESS FOR PRODUCTION OF VIOMYCIN Thomas E. Freaney, Terre Haute, Ind., assignor to Commercial Solvents Corporation, Terre Haute, Ind., a corporation of Maryland No Drawing. Application September 27, 1954 Serial No. 458,678.

Claims. (Cl. 195-109) Viomycin is a recent addition to the list of antibiotic substances. The antibiotic is fully reported and described in U. S. Patent No. 2,633,445, issued March 31, 1953, to William F. Marsh, Rudolph L. Mayer, Robert P. Mull, Caesar R. Scholz and Robert W. Townlcy. It is active against many mycobacteria, staphylococci, and escherichia. This antibiotic has been shown to be highly active in vitro against strains of Mycnbacterium tuberculosis var. hominis H37Rv, Mycobacterium ranae, Bacillus mycoides, Staphylococcus aureus, Escherichia colz', Proteus vulgaris, Diplococcus pneumoniae, Klebsiella pneumoniac, and Bacillus subtilis, and has shown activity in vivo against strains of Staphylococcus aureus and Escherichia coli.

Viomycin is produced by inoculating a nutrient medium with a viable strain of the organism Actinomyces vinaceus. In the above-identified patent, a method for the produc' tion of viomycin is set forth comprising inoculating a medium containing soya peptone, beef extract, dextrose, sodium chloride, and a silicone anti-foaming agent with a spore suspension of Actinomyces vinaceus and incubating the inoculated medium for 120 hours at a temperature of 26 C. while passing sterile air through the medium at a rate of 500 ml. per liter of medium per minute. In the past other nutrient media containing other sources of nitrogen and carbohydrates for the culture of a viomycin producing strain of Actinomyces vinaceus have been employed.

I have now discovered a method for the production of viomycin by the organism Actinomyces vinaceus which results in greatly increased yields over any obtained from previously known or reported methods of production of the antibiotic.

My new process for the production of viomycin consists essentially of cultivating the organism Actinomyces vinaceus-on a liquid nutrient medium aerated at a superficial air velocity of from about 0.7 to about 1.0 until. the pH of the medium reaches 8.0, then increasing the superficial air velocity to from about 1.5 to about 2.0 until the fermentation is completed, and recovering the viomycin produced. The viomycin produced, according to my process can be recovered from the nutrient medium by any convenient process, such as that described in U. S. Patent No. 2,633,445, or in co-pending application Serial No. 370,233, filed July 24, 1953.

The term superficial air velocity, as used herein is defined in the article, Development of a typical aerobic fermentation, by O. G. Wegrich and R. A. Shurter, Ir.,

published in Industrial and Engineering Chemistry, vol- The values defined in the formula are derived as fol- 2,828,245 Patented Mar. 25, 1958 7 2 lows: V, equals the average superficial linear air velocity in feet per minute at the effective pressure, (1 equals the volumetric rate of air flow in standard cubic feet per minute, P equals the effective pressure in pounds per square inch gauge, which is equal to the pressure at the exit of the ferinenter in pounds per square inch absolute 'plus 0.217 times the feet of operating depth above the sparger outlet, D equals the diameter of the fermenter in feet, and T equals the absolute temperature of the fermentation in degrees K.

As the nutrient medium for this fermentation, I can employ any of the nutrient. media known to result 111 the production of viomycin activity. Generally, however, I prefer to use nutrient media containing as a source of assimilable nitrogena soybean product and as a source of carbohydrate, starch or hydrolytic products of starch.

Suitable sources of assimilable nitrogen for the medium include raw soybean meal, solvent extracted soybean meal, soy grits, and soy, flour. Suitable media may be prepared containing from about 1% toabout 6% by weight of the soybean products, but I prefer a medium containing from about 2% to about 3% soybean product as a source of nitrogen.

Suitable sources of carbohydrate for the nutrient medium in my new process include starches, dextrins, dextrose, and malt syrup. I have found that dextrins are'the preferred sources of carbohydrate in the medium. Nutrient media can contain from about 2% to about 7 6% of the carbohydrate source, but I prefer that the media contain from about 2% to about 4% of the carbohydrate source. 7

The organism Actinomyces vinaceus is aerobic, and hence air must be supplied to it. Aeration of the medium during the fermentation may be accomplished by any desired means so long as the air is sterile. A combination of aeration and mechanical agitation of the medium results in more rapid growth of the organism and higher yields of viomycin. The air is preferably introduced through a dispersion unit, or a series of units, which causes the air to be divided into bubbles of small diameter.

Studies of the pH during the course of the fermentation by Actinomyces vinaceus of a'nutrient medium have revealed something of the nature of the changes occurring during the course of the fermentation. The pH of the original medium after sterilization is approximately 6.6. It has been found that the pH undergoes a steady rise during the first hours of the fermentation to a pH of from about 8.0 to about 8.2. At this point, the pH of the fermentation assumes a slight downward trend or a constant level of from a pH of about 7.6 to about 8.0 until the final hours of the fermentation. During the final hours of the fermentation the pH of the fermented medium again assumes a rising trend past a pH of 8.2 to a final pH of about 8.5 at which all antibiotic production appears to cease.

I have now discovered that if the aeration rate, expressed as superficial air velocity, is maintained during the early hours of the fermentation at from about 0.7 to about 1.0 until the pH of the fermenting medium reaches approximately 8.0 and is then maintained at a value of from about 1.5 to about 2.0 until the fermentation is substantially complete, the fermentation process is greatly benefited and yields are realized higher than any previously known or reported. The increase in the superficial air velocity after the initial period of mycelial growth has the effect of reducing the downward trend of the pH, extending the period during which a level pH is maintained, and vastly increasing the yield of viomycin produced.

The agitating means employedfor the fermentation can be of. any of several'standard designs such as, for

example, those of the rake, propeller, or vertical blade:

type... The..type.ofagitationused should bewsuch thatthe organisms are not undulymacerated. Using 3.17 gallons of medium in a -gallon fermenter aerated at a superficial air velocity of 0.7, I have foundzthatuagitationwith vertical blades at a rate of about'200 to: 300-.-R', .P. M;

gives best results.

Aeration and agitation of the mediumgoften cause considerable foaming and in such ev.ent,-::antifoam agents are employed to counteract this veifect. Antifoamgagentsw suitable for use in the production of .viomyein include mineral oils, corn oil, and lard oil.

In carrying out my'new process forgthe:iproductionwof1;. viomycin, the temperature at whichllzeflecbfermentaetion can range from about 25? to 21130111135 1 Tempera-. tures below about 25 C. have an adverse efiect ;.on--the rate of viomycin formation. In other words, .as-;the..tern-; perature is lowered, the fermentation... timezimustzbe-rz lengthened in order to achieve viomycinyieldsrcomparable with those obtained in the fermentation:atrtemperae tures Within the range specified above; .Temperaturesin';

excess of C. adversely alfect the ability of: the organ; ism to produce viomycin and in such casesulow yieldsic of viomycin are obtained regardless-of the.lengthrofftime-; employed for fermentation. Generally, we cpreferrto; grow the organism in a nutrient medium at a temperatureui ranging from about 28 to about 32 C.'"since1at 1hese; temperatures, high yields are obtainedin reasonably shorti periods of time.

The period required to carry fermentation to completion is, of c0urse,'dependent upon numerous factorsasuch as temperature, the nutrient content ofthe---'partic'ularmedium fermented, percentage of inoculu'm,- etc2-" Usually' the fermentation is completewithin about- 5' to- -7 days after inoculation. However, in any given instancethis time may readily be ascertained -by-periodicilassays of the medium to determinethe peak viomycincontent,

The following examples are orfered-toillustrate theimprovement obtained through the use of -my-ifiventionl Example I is offered to show the results obtained when the aeration rate to be employed in the productionof viomycin is determined in the manner customary inthe" fermentationart. InExampl I the results of a series of experiments inwhich various aeration rates were main: tained. throughout the' course ofthe fermentation, are

reported. Example II shows the results of increasing; the aeration rate by various increments 'after;the1pH of the fermenting medium reaches ;a value voffrorn 8.0;

V EXAM-PLE'I V This example sets-forth-the experimental data from which it was calculatedthatthe best single aeration rate for the viomycin fermentation fell 'within- -the range or V from about 0.7 to about 1.0 superficial air velocity."

Three five-gallon fermenters 'each' containing -12yliters of a nutrient medium containing 3% extractedsoy can protein (Nutrisoy) and'2% dextrin were inoculatedwith 1.6% by volume of a pregerrninatedinoculum' of" Actinomyces vinuceus and aerated throughout the period of the fermentation at the rates indicated. The results are set out in Table I below.

In this. experiment,-;,a series of runs' was :condu'ctediim S-gallon. fermenters containing -12 liters.of.;a..medium.,

0 ficial. air velocity toabout 1.5 to about 2.0, until the fermentationis' completed, 1 and recovering the viomycin tation.

containing 3% extracted soybean protein (Nutrisoy), 3% dextrin, and 0.2% Cerelose. The medium was inoculated in the same manner as set forth above with 1.6% by volume of a pregerminated inoculum of Actinomyces vinaceus. Agitation was maintained at the same rate as in the experiment in Example I above, 200 R. P. M. The aeration rate was .varied in two stages, determined by the time at'which the pH'of the fermented medium-first reached 8.0 to 8.2.. The'medium was sampled at 12 hour intervals and when the pH had reached a value of from 8.0 to 8.2 the aeration rate was increased. The aeration rate for the first stage was varied-over a range of from 0.72 to 0.91 superficial air velocity. The rate for the second stage was varied within a range of from 0.36 to 1.63 superficial air velocity. Final viomycin titers were determined after 144 hours of fermentation. The results of this experiment are set out in Table II below.

It is evident from the "data set forth in "Table II abovethat increasingthe superficial 'air'velocity atwhich the viomycin fermentation is aerated after the fermenting medium hasreached-the-pHcf 8.0 to 8.2 has-the-etfect of producing a pronounced increase in the-amount of" viomycin produced during'a normal144 hour-fermentation. This finding is in pronounced contrast-to the indication by the experimental data-asset forth'inEx ample -I above that a superficial air velocity of between"0.7 and- 1.0 is optimum forthep roduction -of viornycin'iif the same aeration rate is maintained throughout the fermen- I intend for my' inventiontobe limited only' by the scope of this specification and'the appended'claims;

Now having describednny invention, what I claim is: i. A process for the productionof-viomycin which" comprises culturing the organism-Ac1tin0mycesyinaceus on a liquid nutrient medium" aerated at a superficial air velocity of from about 0.7. to about 1.0 until the'pH of the medium reaches 8.0; then increasing the superficial air velocity to fronraboutdj "to about 2.0 until the fermentation is completed, and recovering the viomycin produced.

2. The improvement in the process for the production vinqceus on a liquid'nutrient medium which comprises aerating the inoculatedmedium at a superficial air velocity of from about 0.7.to about 1.0 until the pH of thefermenting mediumjeaches. 8.0, then increasing h the superproduced.

3. The improveinent in the process for the production, of 'fviomycin' by culturing the. organism Actin0myces vimzceus on a liquid nutrient .mediumcontaining a soybean product as a source offproteih, and jdextrin asa source of carbohydrate which comprises aerating the inoculated medium at asuperficial air' velocity, of from about 0.7'to about 1.0 until the pH of the-fermenting medium reaches 8.0,f'then increasing ithesuperficial air velocity to from about, 1.5 to. about. 2.0 until the .fermentation is'completed, and'recovering the viomycin produced. V

4. A process for the production of viomycin which comprises culturing theiorganism.Actinomyces vinaceous on a liquid nutrient medium aerated at a superficial air velocity of from about 0.7 to about 1.0 until the pH of the medium reaches 8.0, then increasing the superficial air velocity to from about 1.5 to about 2.0 until the fermentation is completed.

5. In the production of viomycin, wherein a liquid nutrient medium, containing from about 1 to 6% of a soybean product as a source of assimilable nitrogen and from about 2 to 6% of a soluble carbohydrate, is fermented by culturing therein the organism Actinomyces vinaceus, under conditions of aeration, and wherein the pH of the medium, when aerated at a constant rate, increases during the first hours of the fermentation to a pH of from about 8 to 8.2; the improvement which consists substantially in aerating the nutrient medium during said first hours with air introduced at a superficial velocity within the range of 0.7 to 1.0, until the pH of the medium reaches a pH of about 8 to 8.2, then substantially increasing the superficial air velocity to a rate of from about 1.5 to 2 until the fermentation is completed, whereby the yield of viomycin is substantially increased over that obtained by aeration throughout the process at a constant rate.

References Cited in the file of this patent UNITED STATES PATENTS 2,047,669 Cahn July 14, 1936 2,118,370 Wessblad May 28, 1938 2,534,089 Stubbs et a1. Oct. 3, 1950 2,633,445 Marsh et a1 Mar. 31, 1953 OTHER REFERENCES Coffey et al.: Viomycin Productivity of Some Undefined Media, Antibiotics and Chemotherapy, vol. 1, 1951, pages 203-207.

Wegrich et al.: Development of a Typical Aerobic Fermentation, I. and E. Chem., vol. 45, May 1953, pages 1153-1160. 

1. A PROCESS FOR THE PRODUCTION OF VIOMYCIN WHICH COMPRISES CULTURING THE ORGANISM ANTINOMYCES VINACEUS ON A LIQUID NUTRIENT MEDIUM AERATED AT A SUPERFICIAL AIR VELOCITY OF FROM ABOUT 0.7 TO ABOUT 1.0 UNTIL THE PH OF THE MEDIUM REACHES 8.0, THEN INCREASING, THE SUPERFICIAL AIR VELOCITY TO FROM ABOUT 1.5 TO ABOUT 2.0 UNTIL THE FERMENTATION IS COMPLETED, AND RECOVERING THE VIOMYCIN PRODUCED. 